Gastrointestinal lipase inhibitors are molecules of pharmaceutical interest due to their use as anti-obesity drugs. In this study, forty strains isolated from soil and sediments were identified with the ability to produce inhibition of gastrointestinal lipase activity. The biomass extract of these strains showed at least 50% inhibition in the hydrolysis of tributyrin by recombinant Human Pancreatic Lipase (rHPL) or Rabbit Gastric Lipase (RGL) by in vitro assays. Based on gene sequencing, the isolates were identified mainly as Streptomycetes. Moreover, none of the identified strains have been reported to be lipase inhibitor producers, so they can be viewed as potential sources for obtaining new drugs. IC50 values of the three best inhibitor extracts showed that AC104-10 was the most promising strain for production of gastrointestinal lipase inhibitors. AC104-10 shows 99% homology (16S rRNA gene fragment) to Streptomyces cinereoruber strain NBRC 12756. An inhibitory study over trypsin activity revealed that AC104-10 extract, as well as THL, had no significant effect on the activity of this protease, showing its specificity for lipases. In addition, analyzes by MALDI-TOF Mass Spectrometry of the enzyme-inhibitor complex revealed that there is a covalent interaction of the AC104-10 inhibitor with the catalytic serine of the pancreatic lipase, and that the molecular weight of the inhibitor is approximately 686.19 Da.